This application is a U.S. National Stage of International application PCT/FR00/00832, filed Apr. 3, 2000 and published on Oct. 12, 2000 in the French Language.
This invention concerns an apparatus and a method for generating ultrasound of variable power in a receptacle containing a sample to be lysed with or without the presence of glass beads. The frequency range of the ultrasound is between 20 and 50 kilohertz (kHz) approximately.
The background art essentially consists of two methods which use ultrasound to lyse microorganisms in biological applications. The first method involves direct sonication in which the sonotrode is submerged in the sample to be lysed. The principle exploited in this case consists in placing the sonotrode directly in contact with the liquid and generating high energy ultrasound in either a continuous fashion or in pulses to induce intense cavitation within the medium. In general, the sonotrodes used have a very high power output (100 to 1000 Watts [W]) and can be used to lyse a wide variety of different types of sample which are known to be relatively difficult to lyse in a short time (of the order of one minute).
However, two major problems restrict the utility of this method.
Firstly, since the phenomenon of cavitation is very difficult to control, the percentage lysis will not be reproducible across a large number of test runs.
Moreover, high-intensity cavitation induces heavy and uncontrolled nucleic acid fragmentation which can be a problem if the user is trying to detect and amplify in samples which only contain dozens or hundreds of microorganisms per microliter (i.e. very low densities).
Finally, since the sonotrode is in direct contact with the sample, this method cannot be used in the automated analysis of patients"" samples unless a wash step for the said sonotrode is included-such wash steps are time-consuming and costly to implement in automatic analyzers.
There are also other disadvantages related to the above-mentioned problems. Firstly, cavitation induces condensation of part of the sample which further compromises the resolution of the test being performed. Finally, if the acoustic power is too great, excessive heating of the tube can lead to sample breakdown or even melting of the said tube.
The second method involves bath sonication which is fully described in patent U.S. Pat. No. 5,374,522. The principle consists in using an ultrasound cleaning bath filled with water with the bottom of the tubes containing the samples to be lysed submerged therein.
The samples are exposed to an ultrasound field-usually constantxe2x80x94for about 15 minutes (min). Beads present in the tubes are thus induced to move around vigorously which causes them to collide with one another and create shear forces which are sufficient to lyse microorganisms. In contrast to the first method described above, this second method requires the use of glass beads to insure adequately efficient lysis. This second method bypasses certain of the problems mentioned in connection with the preceding direct sonication method.
Thus, with the samples being contained in a sealed tube, nthere is no contamination of the vibrating element since the ultrasound waves are transmitted through the water (which is an excellent medium for the propagation of such waves) from the bottom of the bath into the inside of the sample-containing tube.
Moreover, since the energy density (expressed in terms of Watts per milliliter [W/ml]) generated in the medium is about 300 times lower at the same frequency, there is less cavitation.
The major disadvantage of this method is the fact that it is difficult to automate because of the problems associated with the handling of liquids (the filling and emptying of the bath), because degassing is necessary before sonication and because of the complications after sonication associated with having to manipulate wet tubes (which tend to drip and compromise cleanliness).
There are further disadvantages. Thus, the results are not reproducible because the acoustic power is not the same in all parts of the receptacle placed in the bath for sonication. This is due to the way in which bath sonicators are constructed with one or more transducers below the tank. Thus, the ultrasound field generated by a transducer is not equivalent in all spatial dimensions. As a result, both the efficiency of lysis of microorganisms and the extent of nucleic acid fragmentation vary enormously between different samples, and this variation cannot be controlled.
There are, however, sonication methods based on the sonotrode being in direct contact with the biological sample to be lysed, e.g. Patent Application EP-A-0.337.690.
However, the contact between receptacle and sonotrode is constituted by a planar surface which is not compatible with homogenous lysis throughout the test sample. Such devices do not meet the lysate quality requirements necessary for the release of nucleic acids. The only solution to this is to increase the lysis time considerablyxe2x80x94by a factor of at least twoxe2x80x94with a concomitant increase in the risk of denaturation or even destruction of the nucleic acid molecules which receive the most intense fraction of this heterogeneous sonication.
A comparison of the energy densities (W/ml) gives an idea of the major differences which exist between the two methods of the background art described above. Energy density can be defined as the integral over time of the power delivered to the sample divided by the volume of the sample. This quantity can be experimentally determined by measuring the temperature rise over a given period of time (and the results for the two different methods can be compared).       E    =                  ∫        O        T            ⁢                        W          V                ⁢                  xe2x80x83                ⁢                  ⅆ          t                ⁢                  xe2x80x83                ⁢        where        ⁢                  xe2x80x83                ⁢        E        ⁢                  :                ⁢                  xe2x80x83                ⁢        energy        ⁢                  xe2x80x83                ⁢                  density          .                      
                    ⁢                      xe2x80x83                    ⁢          W                ⁢                  :                ⁢                  xe2x80x83                ⁢        output        ⁢                  xe2x80x83                ⁢        power        ⁢                  xe2x80x83                ⁢                  (          W          )                                xe2x80x83        ⁢          V      ⁢              :            ⁢              xe2x80x83            ⁢      Sample      ⁢              xe2x80x83            ⁢      volume      ⁢              xe2x80x83            ⁢              (        ml        )            
Measurements of the temperature inside the tubes after 15 min of bath sonication give an estimate of the power delivered into the sample:
T0mn=21xc2x0 C.
T15mn=40.6xc2x0 C.
i.e. xcex94t=19.6xc2x0 C. and DW=xcex94txc3x970.3 ml=19.6xc3x970.3=5.88 cal/15 min
therefore Wt=(5.88xc3x974.18)/(60xc3x9715)=0.027 W
The energy density D (in W/ml) in a bath sonicator-type ultrasound bath is:
D=0.027/0.3=0.09 W/ml
It is apparent that the energy density is about three hundred times lower than that generated by a sonotrode directly submerged in the medium since, according to published data, the energy density in this case is generally at least 30 W. This value is confirmed by the data given in document U.S. Pat. No. 5,374,522 on bath sonication.
In accordance with this invention, the proposed apparatus provides a solution to all the above-mentioned problems in that it makes controlled nucleic acid fragmentation possible without broaching the integrity of the tube containing the sample. The entire unit remains perfectly clean because there is no contact between the outer surface of the tube and any liquid, the contents of said tube being entirely isolated from the exterior. This isolation precludes the possibility of any material being ejected out of the tube during sonication, and also considerably reduces evaporation and condensation.
To this effect, this invention concerns an apparatus which includes a sonotrode designed to generate ultrasound of variable power within at least one biological sample containing cells to be lysed, the sample(s) being contained in at least one receptacle suited for that purpose and the sonotrode being in direct contact with the receptacle(s) containing the cells to be lysed without any fluid between the said surfaces (of the sonotrode and of the receptacle), characterized in that the active surface of the sonotrode matches the shape of all or part of each receptacle containing the sample to be lysed; the active surface of the sonotrode which is in contact with the receptacle is concave in shape.
In another embodiment, the active surface of the sonotrode which is in contact with the receptacle is convex in shape.
When the active surface is convex in shape, the surface of the receptacle which adjoins the sonotrode is flexible in such a way that it deforms to fit up tightly against the active surface of said sonotrode.
In all examples, the sonotrode acts cooperatively with at least one glass bead contained in the sample inside a receptacle.
The diameter of each bead is of between 90 and 150 and preferably of 100 micrometers (xcexcm) for the lysis of bacteria, and of between 150 and 1500 and preferably of 100 xcexcm for the lysis of yeast cells. These values are not theoretical and, after extensive research carried out by the applicant, resulted in the submission of patent application PCT/IB98/01475 on Sep. 23, 1998, under French priority of Sep. 23, 1997. The originality of this principle lies in the fact that these values are also applicable to the method of direct sonication.
According to a particularly interesting modified embodiment, the apparatus includes at least two sonotrodes.
Moreover, each receptacle is maintained in physical contact with the sonotrode(s) through a means of pressurization.
Each sonotrode can emit ultrasound at a range of frequencies of between 20 and 50 kHz (more precisely, between 30 and 40 kHz and preferably of close to 35 kHz).
This invention also concerns an ultrasound method for lysing cells in a biological sample contained in a receptacle, which uses at least one sonotrode. According to a first implementation, the method is characterized in that it consists in:
placing the receptacle in direct contact with the active surface of the sonotrode(s), and
activating said sonotrode(s) for long enough to lyse the cells in the sample but preserve the DNA and/or RNA molecules released for subsequent operations, e.g. amplification.
According to a second implementation, the method is characterized in that it consists in:
placing the receptacle in direct contact with the active surface of the sonotrode(s),
activating said sonotrode(s) for long enough to lyse the cells in the sample and fragment the DNA and/or RNA molecules released in such a way that subsequent operations can be performed, e.g. amplification.
According to a modified embodiment of the two preceding implementations, prior to the activation of the sonotrode(s), the receptacle is compressed up against the active surface of said sonotrode(s).
Each sonotrode is activated in the following way:
a sonication time of 10 to 15 minutes,
a cycling ratio of between 40 and 60% (preferably 50%), and
an output power of 10 to 30 W.
According to a preferred mode of implementation of the method, activation of each sonotrode corresponds to the emission of a series of pulses each lasting between 5 and 20 seconds, and preferably between 10 and 15 seconds.